Effects of photobiomodulation (660 nm laser) on anthracycline extravasation: An experimental study

Abstract Objective: to investigate the effect of using different agents (topical hyaluronidase, photobiomodulation, and the association of photobiomodulation with topical hyaluronidase) in preventing the formation of lesions caused by doxorubicin extravasation, as well as in the reduction of lesions formed by extravasation of this drug. Method: a quasi-experimental study conducted with 60 Wistar rats, randomized into four groups with 15 animals each. Group 1 (Control); Group 2 (Hyaluronidase); Group 3 (Photobiomodulation); and Group 4 (Hyaluronidase + Photobiomodulation). A wound was induced by applying 1 mg of doxorubicin to the subcutaneous tissue of the back of the animals. The concentration of topical hyaluronidase was 65 turbidity units/g and the energy employed was 1 joule of 100 mW red laser per square centimeter. With macroscopic evaluation every two days for 28 days, the following variables were observed: skin integrity, presence of blisters, hyperemia, exudate, bleeding, edema, crust, peeling and granulation tissue. Results: the animals from the groups subjected to photobiomodulation obtained better results in the assessment of the following variables: bleeding, hyperemia, exudate, intact skin and edema. Conclusion: it was evidenced that the association of photobiomodulation with topical hyaluronidase was effective in reducing the local effects and assisted in the wound healing process, and that PBM alone was able to prevent appearance of lesions.


Introduction
Extravasation of antineoplastic agents (EA) is the most feared adverse event in Oncology, with the possibility of causing serious injuries, especially when the medication involved is classified as vesicant (1) . When infused outside a vessel, vesicant medications cause bubbles and necrosis.
With a reported incidence in the literature from 0.1% to 6%, its main signs and symptoms are infusion stop, absence of venous return, burning sensation, hyperemia, edema and pain. As it is also considered as an emergency in Oncology, it requires a duly qualified and trained Nursing team for immediate treatment of this event. In view of this, it is necessary to investigate actions towards the prevention of complications referring to extravasation (1)(2) .
One of the most feared vesicant medications is doxorubicin (DOX), an anthracycline binding to Deoxyribonucleic Acid (DNA) that binds to the nucleic acids of tissues, generating free radicals, inhibiting the synthesis of local proteins and leading the tissue to chronic, progressive and increased necrosis, making the lesion deeper, more extensive and painful, exerting direct impacts on the patient's quality of life. It can remain in tissues for up to 28 days, which requires constant evaluation (3) .
Currently, dexrazoxane is the antidote used in DOX extravasation. Its use is approved by the Food and Drugs Administration (FDA) and by the European Commission and has 98% efficacy. The mechanism of action is binding of the drug to the iron molecule preventing the formation of free radicals (4) . In addition to being unfeasible for the public sector (due to its price), it can cause side effects such as nausea, vomiting, mild pain at the infusion site and reversible increases in liver enzymes (2,5) .
Hyaluronidase is a widely used antidote in the extravasation of chemotherapeutics; however, it is specific for non-DNA-binding vesicants (vinca and taxane alkaloids), although it is a potential antidote for anthracyclines. This medication modifies tissue permeability by hyaluronic acid hydrolysis, promoting reabsorption of fluids and reducing the edema. This enzyme is an antidote recommended by the Oncology Nursing Society (ONS), which 1.0 ml subcutaneous application of 150 UI/ml to the extravasation area (1,(6)(7)(8) . After ten minutes since application, an increase in diffusion of the overflowed liquid can be seen in an area from 3 to 5 times greater than in an untreated area.
It is quite used to accelerate the healing process of various types of lesions, promoting improvements in the patients' quality of life and speeding up the treatments (10) .
When applying PBM, photochemical and photophysical reactions occur where excitation of the electrons that release adenosine triphosphate (ATP) used by the cells for development of their functions is stimulated (8,(11)(12)(13) .
For this, it is necessary that light is absorbed by chromophores, which are interrelated molecules (enzymes, cell membranes, extracellular substances) with the ability to absorb light. Cytochrome C Oxidase (CCO) is the main chromophore and is located in unit IV of the mitochondrial respiratory chain. CCO absorption mainly by the red wavelength leads to photodisconnection of nitric oxide (NO), resulting in a reduction of oxidative stress and, consequently, in an increase in the production of reactive oxygen species (ROS), ATP and Ca 2+ ions, favoring the intended biological response (anti-inflammatory, analgesic, scarring) and increasing cell differentiation, proliferation and migration (11,13) .
The association between topical hyaluronidase 65 UTR/g and PBM is already used in an Oncology Outpatient service of a tertiary-level Public Hospital from the inland of the state of São Paulo. However, despite excellent results, the number of patients with extravasation is low, and there is no scientific evidence on this association. Consequently, it was necessary to conduct an experimental study to evidence this practice, thus justifying this study.
Therefore, the objective of this research was to investigate the effect of using different agents (topical

Type of study
An experimental study conducted with 60 albino Wistar adult female rats of the Rattus norvegicus species, aged between 3 and 4 months old. In randomized experimental studies, the researcher randomly assigns the subjects to a control group and to one or more experimental groups, minimizing selection bias, in addition to allowing isolating the effect of an intervention (14) .

Study locus and period
The experiment was carried out from 08/15/2019 to and 90% power will be 15 animals per group (60 animals for all 4 groups).

Study variables
The qualitative variables analyzed were related to the appearance of the extravasation site and to the formation or not of lesions such as: intact skin, blister (intact or ruptured), hyperemia (marginal, central, or generalized), exudate (absent, mild, moderate, severe), bleeding (absent, mild, moderate, severe), edema (absent, mild, moderate, severe), crust (absent and present: loosely adhered, very adhered), peeling (absent or present -mild, moderate, severe) and granulation tissue (present, absent).

Instruments used for data collection
The process of wound formation was monitored by the researcher every 48 hours for 28 days, through an evaluation form that included presence of intact skin, blisters, hyperemia, exudate, bleeding, edema, crust, peeling and granulation tissue (15). At the same time, a database of photographic records (CANON Power Shot SX170IS digital camera with 1/250 speed and distance of 0.7) was prepared to assist in interpreting evolution of the wounds. Angle and distance of the photographs were calculated by a professional photographer from the Botucatu Medical School, in order to standardize the images.

Data collection
After the adaptation period, the animals were

Population
The animals were provided by the Creation Vivarium located in the Biotechnology Institute (Instituto de Biotecnologia, IBTEC) of the "Júlio de Mesquita Filho" State University of São Paulo. Immediately after that, they were kept in the Experimentation Vivarium for a 15-day adaptation period, and only then were they submitted to the experiment. Female rats were selected due to their differences regarding behavior and aggressiveness when compared to males, easing manipulation. Throughout the period, temperature, ammonia level and bed change were controlled, as well as water supply and ad libitum feed. They were clinically examined by a veterinarian at admission to the laboratory and during the entire experiment. In order to ensure environmental enrichment, paper rolls were places in the cages in an attempt to reduce the stress caused by manipulation and isolation.

Selection and randomization criteria
To perform the randomization procedure, after the adaptation period, the animals were identified with numbers from 1 to 60, with a pilot pen in the dorsal region. Four envelopes were prepared and identified with the type of treatment, as follows: Subsequently, one of the laboratory team members made a draw with numbers from 1 to 60.
The first 15 animals drawn were allocated to Envelope 1, and so on.
Each group consisted of 15 animals that were housed in rectangular polypropylene cages with 5 animals each, placed on ALESCO ® ventilated racks, kept under room temperature and with free movement. It is important to note that, after DOX inoculation, the animals were separated, keeping them in individual cages.
The cages were identified with the numbers corresponding to the animals and with the group to which they belonged.

Sample definition
Considering that there is 50% occurrence of necrosis cases control) and assuming that application of the treatment reduces this percentage to zero (no necrosis), the minimum size for carrying out the experiment with a 5% margin of error, 95% compatibility www.eerp.usp.br/rlae

Data treatment and analysis
After data tabulation, a qualitative and descriptive analysis of the variables (bleeding, hyperemia, exudate, bleeding, edema, crust, peeling and granulation tissue) was performed by group (Control, Hyaluronidase, PBM and PBM+H) throughout the treatment days.

Ethical aspects
The research was approved on 02/11/2019 by the

Results
It was observed that the groups that made use of hyaluronidase showed more animal losses (H: 09/15: 60% and PBM+H: 8/15: 53.3%). We believe that this loss can be related to several factors such as: toxicity generated by anthracycline, toxicity by hyaluronidase, and factors related to animal behavior. Although the groups were evaluated every two days, hyaluronidase was administered daily, with the possibility that such manipulation of the animals be considered stressing.
It was noticed that some animals from the PBM group (4/15: 26%) did not show lesions (Figure 1). In animals that presented lesions, it was observed that they started at D4, becoming more evident in group C

Discussion
There was premature death of 23 animals, which can be related to several factors, such as the toxicity generated by anthracycline and hyaluronidase and animal behavior.
It was observed that the animals that died the most were from the groups that contained hyaluronidase. We infer that even when the ointment is being applied correctly to the back of the animal, due to its significant fluidity, it generated ease of licking and possible intoxication. The healing process with epithelial tissue was observed at D12 with animals from the PBM+H group.
It was observed that, after 28 days of evaluation, two animals from the PBM and PBM+H groups showed complete healing of the lesions. At the end of the study, six animals (40%) from the PBM group and two (13%) from the PBM+H presented intact skin ( Figure 5). From photochemical reactions, PBM restores the cellular function through the release of reactive oxygen species, causing cellular balance restoration by the production of antioxidant enzymes and most importantly, without deleterious effects to the organism (4) . Unlike dexrazoxane, which, despite having a proven efficiency of 98%, also causes some side effects such as nausea, vomiting, mild pain at the infusion site and reversible increases in liver enzymes (1,5,23) . Unfortunately, it is unfeasible for many services due to its high cost. This is the first paper to investigate the association of PBM with topical hyaluronidase in DOX extravasation.
However, the efficacy of PBM has already been proven in other types of lesion, even with the possibility of being associated with topical therapies.
PBM is already quite used to heal wounds of various etiologies. In a study conducted to evaluate the effect of laser therapy at different wavelengths on the expression of growth factors and inflammatory mediators in the healing processes of pressure ulcers, it was observed that the wavelength of 658 nm was more effective. It is believed that this effect is associated with inhibition of the inflammatory processes (24) .
In a case study on anthracycline (epirubicin) extravasation in a central venous access (fully implanted catheter) in the right hemithorax, it was observed that the patient showed necrosis in 2/3 of her breast in the first 10 days, lasting for 32 days when she underwent a surgical procedure to dry the necrosis. It is important to report that no antidote was applied to this patient, as 24 hours had already elapsed since extravasation. A skin graft was placed in the site and, after 8 months, the breast reconstruction procedure was performed (25) . A similar result was found in our study regarding the antidote and necrosis formation.
In animals that did not receive the antidote, a greater amount of necrosis was observed at all moments evaluated, starting on the fourth evaluation day and extending steadily and progressively throughout the research; differently from what was found with the animals of the groups that used antidotes. All groups showed necrosis, although to a lesser extent and with improvement of the lesion and disappearance of necrosis at the end of the study.
In the study, we found that the granulation tissue was more evident in the animals from the PBM group, with onset of its appearance at D12, maximum peak at D18, stabilization from D20 and a sharp drop at D26, showing replacement of the granulation tissue by gradual epithelization with consequent wound healing of 2 animals from the PBM group and 2 animals from the PBM+H group.
This study provides diverse evidence for using PBM associated with hyaluronidase in DOX extravasation, providing important and relevant information for the formulation of protocols by Oncology professionals. It Hyaluronidase has been widely used in ophthalmic and dermatological surgeries, as the enzyme degrades the hyaluronic acid matrix, increasing tissue permeability and favoring drug absorption. In addition to that, it is already widely used in EA management, subcutaneously (16) . Although we used a topical presentation in our research, no references to this application route in extravasation were found in the literature, nor on its toxic effects when ingested.
The toxicity generated by anthracycline corresponds to the symptoms observed in the animals, such as diarrhea and weight loss. As it is non-selective, DOX causes various side effects, such as nausea, vomiting, diarrhea and anorexia with consequent weight loss (17) . In addition to these effects, the acute inflammation and oxidative stress caused by DOX can be related to toxicity in multiple organs (18) . We noticed that one animal from the Control Group showed ascites at the end of the study. The autopsy was performed after euthanasia, noticing significant adherence to the liver, spleen, heart and intestines.
Better preservation of skin morphology and reduction of edema and ulcer size were observed in an experimental study with Wistar rats that were administered an intradermal injection of vincristine and were treated with hyaluronidase (6) . This fact corroborates the results found in our study, in which only 2 animals from the PBM+H group presented edema.
PBM also plays an important role in the prevention or reduction of edema during tissue repair processes since, after all, the benefits related to reduction of the edema, oxidative stress and pro-inflammatory cytokines are already well established (19) . This fact is related to a considerable increase in blood flow due to the vasodilating action of nitric oxide (NO); however, at the same time, lymphatic drainage is stimulated, reducing the edema through the control of pro-inflammatory cytokines (20) .
During this research, our study group published a retrospective cross-sectional study of the Oncology

Study limitations
We consider the loss of some animals from the groups in use of hyaluronidase as a limitation of this study, which may have been due to toxicity.
Another limiting factor is the non-existence of literature on photobiomodulation in anthracycline extravasation. We suggest that this technique be encouraged in several Health Institutions and that case reports be published in order to increase the evidence, as it would not be feasible to carry out experimental or clinical studies with extravasations in humans. New technologies should be incorporated in order to ensure better care for cancer patients.

Conclusion
The results evidenced that the association of PBM (660 nm -1 J) with topical hyaluronidase (65 UTR) on DOX extravasation was effective in reducing the local effects and assisted in the wound healing process, as well as that PBM alone was able to prevent the appearance of lesions. This therapy is a good alternative to treat extravasation, with the possibility of being incorporated into the clinical practice.